Polysaccharide therapeutic conjugates

ABSTRACT

A composition includes a polysaccharide at least one retinoid linked to at least one monosaccharide subunit of the polysaccharide with a covalent linkage. The linkage being degradable by hydrolysis during digestion of the composition to provide controlled, delayed, and/or sustained delivery of the at least one retinoid upon enteral administration of the composition to a subject.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/653,940, filed Nov. 28, 2011, the subject matter of which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under EY021126 awardedby The National Institutes of Health. The United States government hascertain rights to the invention.

BACKGROUND

Through its various metabolites, vitamin A, retinol, controls essentialphysiological functions. Both naturally occurring metabolites andretinoid analogues have shown effectiveness in many clinical settingsthat include skin diseases and cancer, and in animal models of humanconditions affecting vision. Retinoid therapies have demonstratedclinical efficacy as treatments for many debilitating diseases.

Typically, retinoids are complexed with soluble proteins that protectthem. These reactive compounds are bound by a number of retinoid-bindingproteins, and are rarely freely solubilized from membranes. Protectionof retinoids also stems from their ability to cluster when esterified bylong-chain fatty acids, and undergo storage in lipid-like droplets inthe liver or as retinosomes in the eye. Evidence that esterification ofretinol and retinol-based drugs within target tissues provides one ofthe most efficient means to improve the absorption and to reduce thetoxicity associated with pharmacological doses of retinoids. Currently,there are few treatments for retinoid deficiency. Thus, there is a needfor compositions and methods of restoring or stabilizing photoreceptorfunction and ameliorating the effects of deficient levels of endogenousretinoids.

SUMMARY

Embodiments described herein relate to polysaccharide therapeuticconjugate compositions that provide controlled release, delayed release,and/or sustained delivery of a therapeutic agent upon enteraladministration of the composition to a subject. The compositions includea polysaccharide and at least one therapeutic agent that is linked to atleast one monosaccharide subunit of the polysaccharide with a covalentlinkage. The linkage is degradable by hydrolysis during digestion of thecomposition to provide controlled release, delayed release, and/orsustained delivery of the therapeutic agent upon enteral administrationof the composition to the subject. In one example, the therapeutic agentcan include a retinoid, and the retinoid is released or delivered at arate effective to provide sustained treatment of an ocular disorder,such as a retinal disease associated with inadequate production of11-cis-retinal.

Other embodiments described herein relate to a method for preparing apolysaccharide therapeutic conjugate composition. The method includesreacting the at least one retinoid with a chlorinating agent to provideat least one retinoid having a primary chloride functional group. The atleast one retinoid having a primary chloride functional group is thenreacted with a monosaccharide subunit of the polysaccharide having acarboxylate functional group in the presence of a phase transfercatalyst. The phase transfer catalyst catalyzes the formation of ahydrolysable carboxylic ester covalent linkage between themonosaccharide subunit and the at least one retinoid.

Further embodiments described herein relate to a method for treating aretinal disease in a subject. The method includes administering to thesubject a therapeutically effective amount of a polysaccharidetherapeutic conjugate composition. The composition includes apolysaccharide and at least one retinoid that is linked to at least onemonosaccharide subunit of the polysaccharide with a covalent linkage.The linkage is degradable by hydrolysis during digestion of thecomposition to provide controlled release, delayed release, and/orsustained delivery of the retinoid upon enteral administration of thecomposition to the subject. In one example, the retinoid is released ordelivered at a rate effective to provide sustained treatment of anocular disorder, such as a retinal disease associated with inadequateproduction of 11-cis-retinal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a generalized structure of an alginic acid blockcopolymer. The block copolymer includes a homopolymeric block ofguluronic acid (G-block), a homopolymeric block of mannuronic acid(M-block) and a block of alternating M and G-residues (MG-block). Alsoillustrated are generalized formulas of a G-block (GGGG), a M-block(MMM) and a MG-block (MGM).

FIG. 2 is a schematic illustration showing a hydrolysis reaction ofalginic acid sodium salt.

FIG. 3 is a schematic illustration showing a synthesis scheme of 9-cisretinal chloride from 9-cis retinol using the chlorinating agent thionyldichloride in solvent.

FIG. 4 is a schematic illustration showing a synthesis scheme of alginicacid-retinoid conjugates using the phase transfer catalyst Aliquat 336in a polar solvent.

FIG. 5 is a schematic illustration showing a synthesis of guluronic acid9-cis retinol conjugate from alginic acid and 9-cis retinal chlorideusing the phase transfer catalyst Aliquat 336 in a polar solvent.

FIG. 6 is schematic illustration showing the synthesis ofchitosan-9-cis-retinol conjugates.

FIGS. 7(A-B) illustrate plots showing ERG response in 5-week-oldLrat^(−/−) mice after oral gavage of chitosan-9-cis-retinol conjugates.

FIGS. 8(A-B) illustrate plots showing in 9-cis-oxime levels in eyes of5-week-old Lrat^(−/−) mice after oral gavage of chitosan-9-cis-retinolconjugates.

FIGS. 9(A-B) illustrate images showing the rod outer segment (ROS) layerof 5-week-old Lrat^(−/−) mice administered a control orchitosan-9-cis-retinol conjugates.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisapplication belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

The terms “therapeutic agent,” “drug” and “active agent” are usedinterchangeably herein to refer to a chemical material or compoundwhich, when administered to an organism (human or animal, generallyhuman) induces a desired pharmacologic effect.

The term “polysaccharide” is intended to include naturally occurringpolysaccharides as well as polysaccharides that are obtained viachemical synthesis or genetic engineering. The term is used to includedisaccharides, oligosaccharides and longer saccharide polymers, whereinthe individual monomeric saccharide units may be naturally occurring ormodified. Modified saccharides include those wherein one or more of thehydroxyl groups are replaced with halogen, aliphatic groups, or arefunctionalized as ethers, amines, phosphates, or the like. Intersugarlinkages within the polysaccharide structure may be α-1,2, α-1,3, α-1,4,α-1,6, β-1,2, β-1,3, β-1,4, β-1,6 linkages, or the like.

By the terms “effective amount” or “pharmaceutically effective amount”of an agent as provided herein are meant a non-toxic but sufficientamount of the agent to provide the desired therapeutic effect. As willbe pointed out below, the exact amount required will vary from subjectto subject, depending on age, general condition of the subject, theseverity of the condition being treated, and the particular active agentadministered, and the like. An appropriate “effective” amount in anyindividual case may be determined by one of ordinary skill in the art byreference to the pertinent texts and literature and/or using routineexperimentation.

By “pharmaceutically acceptable” is meant a carrier comprised of amaterial that is not biologically or otherwise undesirable.

The term “transmembrane” refers to the passage of a substance into orthrough a body membrane, e.g., a mucosal membrane such as thegastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal,corneal, or ocular membranes, so as to achieve a desired therapeutic orprophylactic effect.

The terms “absorption” and “transmembrane absorption” as used hereinrefer to the rate and extent to which a substance passes through a bodymembrane.

The term “controlled release” is intended to refer to any therapeuticagent-containing formulation in which the manner and profile of drugrelease from the formulation are controlled. The term “controlledrelease” refers to immediate as well as nonimmediate releaseformulations, with nonimmediate release formulations including but notlimited to sustained release and delayed release formulations.

The term “delayed release” is used in its conventional sense to refer toa delay in release of a composition from a dosage form following oraladministration, such that the majority of the composition is released inthe lower gastrointestinal (GI) tract. After the dosage form reaches theintended release site, there may or may not be a further mechanismcontrolling the release of the composition from the dosage form.“Delayed release” may thus be an immediate release of all the contentsof a drug dosage form, or it may involve controlled release in asustained manner or in a staged or pulsatile fashion (e.g., when amulti-component device is utilized), wherein the term “sustained” meansthat release occurs during an extended time period, and the terms“staged” and “pulsatile” mean that release occurs in two or more spacedapart pulses.

The terms “pro-drug” and “prodrug” are used interchangeably herein andrefer to any compound, which releases an active parent drug in vivo.Since prodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.)the compounds described herein can be delivered in prodrug form.Prodrugs can be prepared by modifying functional groups present in thecompound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds described herein wherein a hydroxy, amino, sulfhydryl,carboxy, or carbonyl group is bonded to any group that may be cleaved invivo to form a free hydroxyl, free amino, free sulftydryl, free carboxyor free carbonyl group, respectively.

The term “treating” refers to inhibiting a disease, disorder orcondition in a subject, e.g., impeding its progress; and relieving thedisease, disorder or condition, e.g., causing regression of the disease,disorder and/or condition. Treating the disease or condition includesameliorating at least one symptom of the particular disease orcondition, even if the underlying pathophysiology is not affected.

Embodiments described herein relate to polysaccharide therapeuticconjugate compositions that provide controlled release, delayed release,and/or sustained delivery of a therapeutic agent upon enteraladministration of the composition to a subject. The release and/ordelivery of the therapeutic agent is controlled, delayed, and/orsustained in nature, such that the release and/or delivery of thetherapeutic agent from the dosage form is controlled, delayed, and/orsustained after oral administration, and, for example, such that itoccurs in the lower GI tract. That is, controlled, delayed, and/orsustained delivery or release of the therapeutic from the composition ordosage form may occur in a sustained fashion over an extended period oftime (e.g., hours, days, or weeks), or in a staged or pulsatile fashion.

The polysaccharide therapeutic conjugates compositions includetherapeutic agents that are covalently linked to a biodegradablepolysaccharide with a degradable or cleavable linker. The degradable orcleavable linker provides the polysaccharide therapeutic conjugatecompositions described herein with increased thermodynamic stability andabsorption time of the therapeutic agent upon enteral administrationcompared to administration of the therapeutic agent alone. Thepolysaccharide therapeutic conjugate compositions can thus be used ashighly dense therapeutic agents for the treatment of diseases ordisorders where controlled release, delayed release, and/or sustaineddelivery of a therapeutic agent is desired.

In some embodiments, the polysaccharide therapeutic conjugatecomposition includes a polysaccharide and at least one retinoid. The atleast one retinoid can be linked to at least one monosaccharide subunitof the polysaccharide with a covalent linkage. The covalent linkage canbe degradable by hydrolysis or hydrolyzable during digestion of thecomposition to provide controlled, delayed, and/or sustained delivery ofthe retinoid upon enteral administration of the composition to asubject. The controlled, delayed, and/or sustained release can allowtherapeutic levels of retinoid to be maintained in the subject forhouse, days, and/or weeks without the need for constant and/orcontinuous administration of the retinoid.

In some embodiments, the covalent linkage is a hydrolyzable covalentlinkage, e.g., an acid-labile linkage, that is cleavable underphysiological conditions of the lower gastrointestinal (GI) tract, e.g.,intestines, to provide controlled, delayed, and/or sustained delivery orrelease of the retinoid to treat an ocular disorder, such as a retinaldisease associated with inadequate production of 11-cis-retinal.

An example of a hydrolysable covalent linkage that slowly degrades orhydrolyzes during digestion of the composition includes a hydrolysableacrylate, ester, ether, thioether, disulfide, amide, imide secondaryamine, direct carbon (C—C), carboxylic ester, sulfate ester, sulfonateester, phosphate ester, urethane, and/or carbonate covalent linkage. Incertain embodiments, the hydrolysable covalent linkage can include ahydrolysable carboxylic ester linkage. For example, carboxylic esterlinkages refer to a structure of either:

wherein Poly and Ret denote a monosaccharide subunit of a polysaccharideand a retinoid respectively.

The retinoid and the polysaccharide can be covalently linked directly orwith a linker or spacer. The spacer can include alkyl or aryl chains,such as an alkyl group, an alkenyl group, an alkynyl group, an arylgroup. In some embodiments, the alkyl group is a C₁-C₁₅, C₁-C₁₀, C₁-C₅,or a C₁-C₃ alkyl, alkenyl, alkynyl, or aryl group. In some embodiments,a spacer can include two or more functional groups allowing forhydrolysable covalent bonds to a monosaccharide subunit of apolysaccharide and to the retinoid.

In this case, hydrolysis of the bond between the retinoid and the spacerwill release the therapeutic, while, hydrolysis of the bond between thespacer and the polysaccharide will provide a prodrug which will not beactive until the linkage or bond between the retinoid and linkingcompound is hydrolyzed. Providing a prodrug in this manner may beadvantageous in certain controlled, delayed, and/or sustained releaseapplications.

The polysaccharide of the polysaccharide therapeutic conjugatecomposition can include any polysaccharide having at least onemonosaccharide subunit capable of forming a covalent linkage with aretinoid as described herein. In some embodiments, the polysaccharide isa water-soluble polymer that is capable of forming hydrolysable covalentlinkages with multiple molecules of one or more retinoids per moleculeof polysaccharide thereby forming polysaccharide-retinoid conjugates.

In some embodiments, the monosaccharide subunit capable of forming acovalent linkage with a retinoid is a monosaccharide compound containinguronic acids or uronic acid derivatives. A uronic acid has a carboxylicgroup (—COOH) on the terminal carbon that is not part of the sugar ringstructure subunit making the carboxylic group available to form acovalent linkage with a retinoid as described herein. Examples of uronicacids and uronic acid derivatives include galacturonic acid, glucuronicacid, mannuronic acid, their lactones, their esters, and their amides,which can be produced by known methods.

In some embodiments, the polysaccharide can include polymers comprisedof uronic acid monosaccharide subunits. In some embodiments, thepolysaccharide can include alginic acids (also known as algin oralginate). Alginic acids occur as linear copolymers of (1-4)-linkedβ-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues,respectively, covalently linked together in different sequences orblocks referred to as “block copolymers”. As illustrated in FIG. 1, themonomers can appear in homopolymeric blocks of consecutive G-residues(G-blocks), consecutive M-residues (M-blocks) or alternating M andG-residues (MG-blocks). For example, a block polymer of aginic acid oranother polysaccharide can be referred to as a “diblock copolymer” if itcontains two different homopolymeric (G or M) or alternating (MG)blocks. Triblocks, tetrablocks, multiblocks, etc. can also be made.

Alginic acid is a natural acidic polysaccharide extracted from so calledbrown algae (Phaecophyceae) with a high molecular weight varying betweenabout 30,000 and 200,000 or higher, and containing chains formed byD-mannuronic acid and L-guluronic acid. The degree of polymerizationvaries according to the type of alga used for extraction, the season inwhich the algae were gathered and the place of origin of the algae, aswell as the age of the plant itself. The main species of brown algaeused to obtain alginic acid are, for example, Macrocystis pyrifera,Laminaria cloustoni, Laminaria hyperborea, Laminaria flexicaulis,Laminaria digitata, Laminaria japonica, Ascophyllum nodosum, Lessoniaflavicans, Durvillea antartica, Ecklonia maxima and Fucus serratus. Itis also produced by two bacterial genera Pseudomons and Azotobacter.

The alginic acid to be used as a starting material may be anycommercially available alginic acid. For the case in which polyguluronicacids are the main target products, an alginic acid rich in G blocks ispreferred. Alginic acids extracted from the seaweeds Laminariahyperborea and Lessonia flavicans are particularly rich in G-blocks. Forthe case in which polymannuronic acids are the main target products, analginic acid rich in M blocks is preferred. Alginic acids extracted fromthe seaweeds Laminaria japonica and Durvillea antartica are particularlyrich in M-blocks. For the case in which random polymers containingguluronic acid and mannuronic acid are the main target products, analginic acid rich in MG blocks is preferred. Alginic acids extractedfrom the seaweed Ecklonia maxima are particularly rich in MG-blocks.Alternatively, synthetically prepared alginates having a selected M andG unit proportion and distribution prepared by synthetic routes, such asthose analogous to methods known in the art, can be used.

In some embodiments, it may be advantageous to create smaller blockunits of alginate prior to synthesizing retinoid-alginate conjugates.Therefore, the alginate material may be treated to provide a material oflower molecular weight, particularly at or below the renal threshold forclearance by humans. In some embodiments, the alginate or any otherpolysaccharide is reduced to a molecular weight of about 1000 to about80,000 daltons, for example, about 1000 to about 60,000 daltons. Thereduction in molecular weight can be effected by hydrolysis under acidicconditions or by oxidation, to provide the desired molecular weight. Inan exemplary embodiment illustrated in FIG. 2, an alginic acid sodiumsalt starting material can be hydrolyzed by hydrochloric acid in asodium bicarbonate buffer to create smaller copolymer block units.

In other embodiments, the polysaccharide can include chitin or chitosan.Chitosan is a linear polysaccharide composed of randomly distributedβ-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit). Chitosan can be made by, forexample, treating shrimp and other crustacean shells with the alkalisodium hydroxide. Synthesis of chitosan-retinoid conjugates can beprepared from the chitosan as shown, for example, in FIG. 6. In someembodiments, the chitosan can have a molecular weight of about 1000 toabout 80,000 daltons, for example, about 1000 to about 60,000 daltons orabout 3000 to about 10,000 daltons.

The retinoid that is used in the polysaccharide therapeutic conjugatecomposition can include any retinoid having a functional group capableof forming a covalent linkage with a monosaccharide subunit of apolysaccharide as described herein. Examples of retinoids can includevitamin A (retinol), a known physiologically active metabolite thereofand their synthetic derivatives. Physiologically active metabolites ofretinol can include 11-cis-retinal, the visual chromophore,all-trans-retinoic acid, the 9-cis-isomer of all-trans-retinoic acid,and the retro-retinoids, anhydroretinol (AR) and14-hydroxy-4,14-retro-retinol (14-HRR) andall-trans-13,14-dihydroxy-retinol.

In some embodiments, the retinoid is a retinal derivative, such as asynthetic retinal derivative. Synthetic retinal derivatives can includederivatives of 9-cis-retinal or 11-cis-retinal in which the aldehydicgroup in the polyene chain is converted to an ester, ether, alcohol,hemiacetal, acetal, oxime, as further described herein. Such syntheticretinal derivatives include 9-cis-retinyl esters, 9-cis-retinyl ethers,9-cis-retinol, 9 cis-retinal oximes, 9-cis-retinyl acetals,9-cis-retinyl hemiacetals, 11-cis-retinyl esters, 11-cis-retinyl ethers,11-cis-retinol, 11-cis-retinyl oximes, 11-cis-retinyl acetals and 11-cisretinyl hemiacetals, as further described herein. The synthetic retinalderivative once conjugated to a polysaccharide can be controllablyreleased from the polysaccharide-retinoid conjugate during digestionupon enteral administration to provide a natural or synthetic retinal,such as for example, 9-cis-retinal, 11-cis-retinal or a syntheticretinal analog thereof, such as those described herein or inInternational Application No. PCT/US04/07937, filed Mar. 15, 2004, (thedisclosure of which is incorporated by reference herein).

An example of a synthetic retinal derivative can include a retinylester. In some embodiments, the retinyl ester is a 9-cis-retinyl esteror an 11-cis-retinyl ester. The ester substituent can include, forexample, a carboxylic acid, such as a mono- or polycarboxylic acid. Asused herein, a “polycarboxylic acid” is a di-, tri- or higher ordercarboxylic acid. In some embodiments, the carboxylic acid is a C₁-C₂₂,C₂-C₂₂, C₃-C₂₂, C₁-C₁₀, C₂-C₁₀, C₃-C₁₀, C₄-C₁₀, C₄-C₈, C₄-C₆ or C₄monocarboxylic acid, or polycarboxylic acid.

Examples of carboxylic acids include acetic acid, propionic acid,butyric acid, valeric acid, caproic acid, caprylic acid, pelargonicacid, capric acid, lauric acid, oleic acid, stearic acid, palmitic acid,myristic acid or linoleic acid. The carboxylic acid also can be, forexample, oxalic acid (ethanedioic acid), malonic acid (propanedioicacid), succinic acid (butanedioic), fumaric acid (butenedioic acid),malic acid (2-hydroxybutenedioic acid), glutaric acid (pentanedioicacid), adipic acid (hexanedioic acid), pimelic acid (heptanedioic),suberic acid (octanedioic), azelaic acid (nonanedioic acid), sebacicacid (decanedioic acid), citric acid, oxaloacetic acid, ketoglutaraticacid, or the like.

In some embodiments, the retinyl ester is a 9-cis-retinyl ester or an11-cis-retinyl ester including a C₃-C₁₀ polycarboxylic acid substituent.(In this context, the terms “substituent” or “group” refer to a radicalcovalently linked to the terminal oxygen in the polyene chain). Inanother embodiment, the retinyl ester is a 9-cis-retinyl ester or an11-cis retinyl ester including a C₂-C₂₂ or C₃-C₂₂ polycarboxylic acidsubstituent. The polycarboxylic acid substituent can be, for example,succinate, citrate, ketoglutarate, fumarate, malate or oxaloacetate. Inanother embodiment, the retinyl ester is a 9-cis-retinyl ester or an 11cis-retinyl ester including a C₃-C₂₂ di-carboxylic acid (di-acid)substituent. In some embodiments, the polycarboxylic acid is not9-cis-retinyl tartarate or 11-cis-retinyl tartarate. In someembodiments, the retinyl ester is not a naturally occurring retinylester normally found in the eye. In some embodiments, the retinyl esteris an isolated retinyl ester. As used herein, “isolated” refers to amolecule that exists apart from its native environment and is thereforenot a product of nature. An isolated molecule may exist in a purifiedform or may exist in a non-native environment.

In another example, the retinal derivative can be a 9-cis-retinyl esteror ether of the following formula I:

In some embodiments, A is CH₂OR, where R can be an aldehydic group, toform a retinyl ester. An aldehydic group can be a C₁ to C₂₄ straightchain or branched aldehydic group. The aldehydic group can also be a C₁to C₁₄ straight chain or branched aldehydic group. The aldehydic groupcan be a C₁ to C₁₂ straight chain or branched aldehydic group, such as,acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, hexanal,heptanal, octanal, nonanal, decanal, undecanal, dodecanal. R can be a C₁to C₁₀ straight chain or branched aldehydic group, a C₁ to C₈ straightchain or branched aldehydic group, or a C₁ to C₆ straight chain orbranched aldehydic group.

R can further be a carboxylate group of a dicarboxylic acid or othercarboxylic acid (e.g., a hydroxyl acid) to form a retinyl ester (some ofwhich are also referred to as retinoyl esters). The carboxylic acid canbe, for example, oxalic acid (ethanedioic acid), malonic acid(propanedioic acid), succinic acid (butanedioic), fumaric acid(butenedioic acid), malic acid (2-hydroxybutenedioic acid), glutaricacid (pentanedioic acid), adipic acid (hexanedioic acid), pimelic acid(heptanedioic), suberic acid (octanedioic), azelaic acid (nonanedioicacid), sebacic acid (decanedioic acid), citric acid, oxaloacetic acid,ketoglutaratic acid, or the like.

R can also be an alkane group, to form a retinyl alkane ether. Examplesof alkane groups include, C₁ to C₂₄ straight chain or branched alkyls,such as, methane, ethane, butane, isobutane, pentane, isopentane,hexane, heptane, octane or the like. For example, the alkane group canbe a linear, iso-, sec-, tert- or other branched lower alkyl rangingfrom C₁ to C₆. The alkane group also can be a linear, iso-, sec-, tert-or other branched medium chain length alkyl ranging from C₈ to C₁₄. Thealkane group also can be a linear, iso-, sec-, tert- or other branchedlong chain length alkyl ranging from C₁₆ to C₂₄.

R can further be an alcohol group, to form a retinyl alcohol ether.Examples of alcohol groups include linear, iso-, sec-, tert- or otherbranched lower alcohols ranging from C₁ to C₆, linear, iso-, sec-, tert-or other branched medium chain length alcohols ranging from C₈ to C₁₄,or linear, iso-, sec-, tert- or other branched long chain length alkylranging from C₁₆ to C₂₄.

The alcohol group can be, for example, methanol, ethanol, butanol,isobutanol, pentanol, hexanol, heptanol, octanol, or the like.

R can also be a carboxylic acid, to form a retinyl carboxylic acidether. Example of alcohol groups are linear, iso-, sec-, tert- or otherbranched lower carboxylic acids ranging from C₁ to C₆, linear, iso-,sec-, tert- or other branched medium chain length carboxylic acidsranging from C₈ to C₁₄, or linear, iso-, sec-, tert- or other branchedlong chain length carboxylic acids ranging from C₁₆ to C₂₄. Examples ofcarboxylic acid groups are example, acetic acid, propionic acid, butyricacid, valeric acid, caproic acid, caprylic acid, pelargonic acid, capricacid, lauric acid, oleic acid, stearic acid, palmitic acid, myristicacid, linoleic acid, succinic acid, fumaric acid or the like.

The retinyl derivative can be a retinyl hemiacetal, where A is CH(OH)OR.R can be any of the R groups set forth above in Formula I. R istypically a lower alkane, such as a methyl or ethyl group, or a C₁ to C₇saturated and unsaturated, cyclic or acyclic alkane, with or withouthetero atoms, as described herein.

The retinyl derivative can be a retinyl acetal, where A isCH(OR_(a))OR_(b). Each of R_(a) and R_(b) can be independently selectedfrom any of the R groups set forth above in Formula I. R_(a) and R_(b)are typically a C₁ to C₇ saturated and unsaturated, cyclic or acyclicalkane, with or without hetero atoms, as described herein.

The retinyl derivative can also be a retinyl oxime, where A is CH:NOH orCH:NOR. R can be any of the R groups set forth above in Formula I. R istypically a hydrogen, or an alkane.

Examples of synthetic retinal derivatives include 9-cis-retinyl acetate,9-cis-retinyl formate, 9-cis-retinyl succinate, 9-cis-retinyl citrate,9-cis-retinyl 35 ketoglutarate, 9-cis-retinyl fumarate, 9-cis-retinylmalate, 9-cis-retinyl oxaloacetate, 9-cis-retinal oxime, 9-cis-retinalO-methyl oximes, 9-cis-retinal O-ethyl oximes, and 9-cisretinal methylacetals and hemi acetals, 9-cis-retinyl methyl ether, 9-cis-retinylethyl ether, and 9-cis-retinyl phenyl ether.

In a related embodiment, the retinal derivative can be an 11-cis-retinylester or ether of the following formula II:

A can be any of the groups set forth above in Formula I.

Examples of synthetic retinal derivatives include, 11-cis-retinylacetate, 11-cis-retinyl formate, 11-cis-retinyl succinate,11-cis-retinyl citrate, 11-cis-retinyl ketoglutarate, 11-cis-retinylfumarate, 11-cis-retinyl malate, 11-cis-retinal oxime, 11-cis-retinalO-methyl oxime, 11-cis-retinal O-ethyl oximes and 11-cis-retinal methylacetals and hemi acetals, 11-cis-retinyl methyl ether, 11-cis-retinylethyl ether.

In additional embodiments, the synthetic retinal derivatives can be, forexample, a derivative of a 9-cis-retinyl ester, a 9-cis-retinyl ether,an 11-cis-retinyl ester or an 11-cis-retinyl ethers, such as, an acyclicretinyl ester or ethers, a retinyl ester or ether with a modifiedpolyene chain length, such as a trienoic or tetraenoic retinyl ester orether; a retinyl ester or ether with a substituted polyene chain, suchas alkyl, halogen or heteratom-substituted polyene chains; a retinylester or ether with a modified polyene chain, such as a transorcis-locked polyene chain, or with, for example, allene or alkynemodifications; and a retinyl ester or ether with a ring modification(s),such as heterocyclic, heteroaromatic or substituted cycloalkane orcycloalkene rings.

The synthetic retinal derivative can be a retinyl ester or ether of thefollowing formula III:

A can be any of the groups set forth above for formula (I). R₁ and R₂can be independently selected from linear, iso-, sec-, tert- and otherbranched alkyl groups as well as substituted alkyl groups, substitutedbranched alkyl, hydroxyl, hydroalkyl, amine, amide, or the like. R₁ andR₂ can independently be lower alkyl, which means straight or branchedalkyl with 1-6 carbon atom(s) such as methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, pentyl, hexyl, or the like. Examples ofsubstituted alkyls and substituted branch alkyls include alkyls,branchedalkyls andcyclo-alkyls substituted with oxygen, hydroxyl,nitrogen, amide, amine, halogen, heteroatom or other groups. Examples ofheteroatoms include sulfur, silicon, and fluoro- or bromosubstitutions.

R₁ or R₂ also can be a cyclo-alkyl, such as hexane, cyclohexene, benzeneas well as a substituted cycloalkyl. Suitable substituted cyclo-alkylsinclude, for example, cyclo-alkyls substituted with oxygen, hydroxyl,nitrogen, amide, amine, halogen, heteroatom and/or other groups.Suitable heteroatoms include, for example, sulfur, silicon, and fluoro-or bromo-substitutions.

The synthetic retinal derivative can also have a modified polyene chainlength, such as the following formula IV:

A can be any of the groups set forth above for formula (I). The polyenechain length can be extended by 1, 2, or 3 alkyl, alkene or alkylenegroups. According to formula (IV), each n and n_(j) can be independentlyselected from 1, 2, or 3 alkyl, alkene or alkylene groups, with theproviso that the sum of the n and n_(j) is at least 1.

The synthetic retinal derivative can also have a substituted polyenechain of the following formula V:

A can be any of the groups set forth above for formula (1). Each of R₁to R₈ can be independently selected from hydrogen, alkyl, branchedalkyl, cyclo-alkyl, halogen, a heteratom, or the like. Examples ofalkyls include methyl, ethyl, propyl, substituted alkyl (e.g., alkylwith hydroxyl, hydroalkyl, amine, amide) or the like. Examples ofbranched alkyls are isopropyl, isobutyl, substituted branched alkyl, orthe like. Examples of cyclo-alkyls are cyclohexane, cycloheptane, andother cyclic alkanes as well as substituted cyclic alkanes, such assubstituted cyclohexane or substituted cycloheptane. Examples ofhalogens are bromine, chlorine, fluorine, or the like. Examples ofheteroatoms are, sulfur, silicon, and fluoro- or bromo-substitutions.Examples of substituted alkyls, substituted branch alkyls andsubstituted cyclo-alkyls include, alkyls, branched alkyls andcyclo-alkyls substituted with oxygen, hydroxyl, nitrogen, amide, amine,halogen, heteroatom or other groups.

For example, the synthetic retinal derivative can be selected from thefollowing: a 9-ethyl-11-cis-retinyl ester, ether, oxime, acetal orhemiacetal; a 7-methyl-11-cis-retinyl ester, ether, oxime, acetal orhemiacetal; a 13-desmethyl-llcis-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis10-F-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-10-C₁-retinyl ester, ether, oxime, acetal orhemiacetal; an ll-cis-10-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-10-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-F-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-C₁-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-12-F-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-12-C₁-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-12 methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis 10-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-12-F-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-12-C₁-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-12-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-14-F-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-14-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-14-ethylretinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-14-Fretinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-14 methyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-14-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; or the like.

The synthetic retinal derivative further can have a modified ringstructure. Examples include, derivatives containing ring modifications,aromatic analogs and heteroaromatic analogs of the following formulaeVI, VII and VIII, respectively:

A can be any of the groups set forth above for formula (I). Each of R₁to R₆, as applicable, can be independently selected from hydrogen,alkyl, substituted alkyl, hydroxyl, hydroalkyl, amine, amide, halogen, aheteratom, or the like. Examples of alkyls are methyl, ethyl, propyl,isopropyl, butyl, isobutyl or the like. Examples of halogens arebromine, chlorine, fluorine, or the like. Examples of heteroatoms aresulfur, silicon, or nitrogen. In formulae VII, X can be, for example,sulfur, silicon, nitrogen, fluoro- or bromo substitutions. Similarly,9-cis-synthetic retinal derivatives containing ring modifications,aromatic analogs and heteroaromatic analogs of those shown in formulaeVI, VII and VIII are contemplated.

The synthetic retinal derivative also can have a modified polyene chain.Examples of derivatives include those with a trans/cis lockedconfiguration, 6s-locked analogs, as well as modified allene, alkene,alkyne or alkylene groups in the polyene chain. In one example, thederivative is an 11-cis-locked analog of the following formula IX:

A can be any of the groups set forth above for formula (1). R₃ can be,for example, hydrogen, methyl or other lower alkane or branch alkane. ncan be 0 to 4. m plus 1 equals 1, 2 or 3.

In one embodiment, the synthetic retinal derivative can be an11-cis-locked analog of the following formula X:

n can be 1 to 4. A can be any of the groups set forth above for formula(I).

The synthetic retinal derivative is a 9,11,13-tri-cis-7-ring retinylester or ether, an 11,13-di-cis-7-ring retinyl ester or ether, an11-cis-7-ring retinyl ester or ether or a 9,11-di-cis-7-ring retinylester or ether.

In another example, the synthetic retinal derivative is a 6s-lockedanalog of formula XI. A can be any of the groups set forth above forformula (1). R₁ and R₂ can be independently selected from hydrogen,methyl and other lower alkyl and substituted lower alkyl. R₃ can beindependently selected from an alkene group at either of the indicatedpositions.

The synthetic retinal derivative can be a 9-cis-ring-fused derivative,such as those shown in formulae XII-XIV. A can be any of the groups setforth above for formula (I). The synthetic retinal derivative also canbe of the following formula XV or XVI.

The synthetic retinal derivative also can be of the following formula XVor XVI.

A can be any of the groups set forth above for formula (1). Each of R₂to R₅, R₇ to R₁₄, R₁₆ and R₁₇ can be absent or independently selectedfrom hydrogen, alkyl, branched alkyl, halogen, hydroxyl, hydroalkyl,amine, amide, a heteratom, or the like. Alkyls include, for example,methyl, ethyl, propyl, substituted alkyl (e.g., alkyl with hydroxyl,hydroalkyl, amine, amide), or the like. Branched alkyls can be, forexample, isopropyl, isobutyl, substituted branched alkyl, or the like.Halogens include, for example, bromine, chlorine, fluorine, or the like.Heteroatoms include, for example, sulfur, silicon, and fluoro- or bromosubstitutions. Substituted alkyls and substituted branch alkyls include,for example, alkyls and branched alkyls substituted with oxygen,hydroxyl, nitrogen, amide, amine, halogen, heteroatom or other groups.Each of n and n₁ can be independently selected from 1, 2, or 3 alkyl,alkene or alkylene groups, with the proviso that the sum of the N and n₁is at least 1. In addition, R₃-R₄ and/or R₂-R₁₆ can comprise an alkenegroup in the cyclic carbon ring, in which case R₁₇ is absent. R₁₀ andR₁₃ together can form a cycloalkyl, such as a five, six, seven or eightmember cyclo-alkyl or substituted cyclo-alkyl, such as, for example,those shown in Formulae IX, X, XII, XIII and XIV.

Methods of making synthetic retinals and derivatives are disclosed in,for example, the following references: Anal. Biochem. 272:232-42 (1999);Angew. Chem. 36:2089-93 (1997); Biochemistry 14:3933-41 (1975);Biochemistry 21:384-93 (1982); Biochemistry 28:2732-39 (1989);Biochemistry 33:408-16 (1994); Biochemistry 35:6257-62 (1996);Bioorganic Chemistry 27:372-82 (1999); Biophys. Chem. 56:31-39 (1995);Biophys. J. 56: 1259-65 (1989); Bio-phys. J. 83:3460-69 (2002);Chemistry 7:4198-204 (2001); Chemistry (Europe) 5:1172-75 (1999); FEBS158:1 (1983); J. Am. Chem. Soc. 104:3214-16 (1982); J. Am. Chem. Soc.108:6077-78 (1986); J. Am. Chem. Soc. 109:6163 (1987); J. Am. Chem. Soc.112:7779-82 (1990); J. Am. Chem. Soc. 119:5758-59 (1997); J. Am. Chem.Soc. 121:5803-04 (1999); J. American Chem. Soc. 123:10024-29 (2001); J.American Chem. Soc. 124:7294-302 (2002); J. Biol. Chem. 276:26148-53(2001); J. Bioi. Chem. 277:42315-24 (2004); J. Chem. Soc.-Perkin T1:1773-77 (1997); J. Chem. Soc.-Perkin T 1:2430-39 (2001); J. Org. Chem.49:649-52 (1984); J. Org. Chem. 58:3533-37 (1993); J. Physical ChemistryB 102:2787-806 (1998); Lipids 8:558-65; Photochem. Photobiol. 13:259-83(1986); Photochem. Photobiol. 44:803-07 (1986); Photochem. Photobiol.54:969-76 (1991); Photochem. Photobiol. 60:64-68 (1994); Photochem.Photobiol. 65:1047-55 (1991); Photochem. Photobiol. 70:111-15 (2002);Photochem. Photobiol. 76:606 615 (2002); Proc. Natl. Acad. Sci. USA88:9412-16 (1991); Proc. Natl. Acad. Sci. USA 90:4072 76 (1993); Proc.Natl. Acad. Sci. USA 94:13442-47 (1997); and Proc. R. Soc. Lond. SeriesB, Biol. Sci. 233(1270): 55-76 (1988) (the disclosures of which areincorporated by reference herein).

Retinyl esters can be formed by methods known in the art such as, forexample, by acid-catalyzed esterification of a retinol with a carboxylicacid, by reaction of an acyl halide with a retinol, bytransesterification of a retinyl ester with a carboxylic acid, byreaction of a primary halide with a carboxylate salt of a retinoic acid,by acid-catalyzed reaction of an anhydride with a retinol, or the like.In an example, retinyl esters can be formed by acid-catalyzedesterification of a retinol with a carboxylic acid, such as, aceticacid, propionic acid, butyric acid, valerie acid, caproic acid, caprylicacid pelargonic acid, capric acid, lauric acid, oleic acid, stearaticacid, palmitic acid, myristic acid, linoleic acid, succinic acid,fumaric acid or the like. In another example, retinyl esters can beformed by reaction of an acyl halide with a retinol (see, e.g., VanHooser et al., Proc. Natl. Acad. Sci. USA, 97:8623-30 28 (2000)).Suitable acyl halides include, for example, acetyl chloride or the like.

Retinyl ethers can be formed by methods known in the art, such as forexample, reaction of a retinol with a primary alkyl halide.

In some embodiment, the retinoid can include a trans-retinoid or acis-retinoid. Trans-retinoids can be isomerized to cis-retinoids byexposure to UV light. For example, all-trans-retinal, all-transretinol,all-trans-retinyl ester or all-trans-retinoic acid can be isomerized to9-cis-retinal, 9-cis-retinol, 9-cis-retinyl ester or 9-cis-retinoicacid, respectively. Trans-retinoids can be isomerized to 9-cis-retinoidsby, for example, exposure to a UV light having a wavelength of about 365nm, and substantially free of shorter wavelengths that cause degradationof cis-retinoids, as further described herein.

Retinyl acetals and hemiacetals can be prepared, for example, bytreatment of 9-cis- and 11-cis-retinals with alcohols in the presence ofacid catalysts. Water formed during reaction is removed, for example byAl₂O₃ of a molecular sieve.

Retinyl oximes can be prepared, for example, by reaction of a retinalwith hydroxylamine, O-methyl- or O-ethylhydroxylamine, or the like. Insome embodiments, a retinoid can further include a transition stateinhibitor of RPE65. For example, the amide ester retinylamide.

In some embodiments, the carboxylate anion of a polysaccharide monomersubunit can act as an alkylating agent with a retinoid having an alkylto form covalent ester linkages. In the case that an alkyl chloride isused, an iodide salt can catalyze the reaction. The carboxylate salt maybe generated in situ from a carboxylic acid. This reaction can sufferfrom anion availability problems and, therefore, can benefit from theaddition of phase transfer catalysts and/or highly polar aproticsolvents such as dimethylformamide (DMF) or dichloromethane (DCM).

Therefore, in another embodiment, a method for preparing a compositionwhich comprises a polysaccharide covalently linked to at least oneretinoid includes reacting the at least one retinoid with a chlorinatingagent to provide at least one retinoid having a primary chloridefunctional group. The method further includes reacting the resultantretinoid chloride with a monosaccharide subunit of the polysaccharidehaving a carboxylate functional group in the presence of a phasetransfer catalyst. The phase transfer catalyst catalyzes the formationof a hydrolysable carboxylic ester covalent linkage between themonosaccharide subunit and the retinoid. An example of a phase transfercatalyst for use in the present invention can include a quaternaryammonium salt, such as Aliquat 336 (also known as Starks' catalyst).

In the presence of a phase transfer catalyst, a retinoid having aterminal alkyl chloride can form a carboxylic ester covalent bond withthe carboxylate ion of either a β-D-mannuronate (M) or α-L-guluronate(G) subunit of an alginate polymer. Thus, in some embodiments, retinoidswith a primary halide group can be covalently linked directly to acaboxylate ion of a monosaccharide subunit. In an exemplary embodiment,illustrated in FIG. 4, a 9-cis retinal chloride is conjugated toalginate in the presence of Aliquat 336 phase transfer catalyst andsolvent solution through a carboxylic ester linkage.

However, because few retinoids have halide groups, the more applicablemeans of providing a degradeable covalent linkage is to first halogenatea retinoid with a holgenating agent to synthesize a correspondingretinal halide. For example, alkyl halides can be prepared by reactingalcohols with a halogenating agent. An exemplary method includesreacting a retinoid having a terminal hydroxyl functional group (e.g.,9-cis retinol, 11-cis retinol or derivatives thereof), with achlorinating agent to synthesize a corresponding retinal halide (9-cisretinal chloride, 11-cis retinal chloride or derivatives thereof). Theterminal —OH functional group can be converted into an alkyl chloride byreacting the —OH functional group with a chlorinating agent using aninternal nucleophilic substitution reaction. In each case the —OHfunctional group reacts first as a nucleophile, attacking theelectrophilic center of the chlorinating agent. A displaced chloride ionthen completes the substitution displacing the leaving group.

An example of a chlorinating agent can include thionyl chloride (SOCl₂),phosphorous trichloride (PCl₃), or phosphorous pentachloride (PCl₅). Incertain embodiments, thionyl chloride is especially convenient, becausethe byproducts are gaseous. In an exemplary embodiment illustrated inFIG. 3, 9-cis retinal chloride can be synthesized from 9-cis retinolusing the chlorinating agent thionyl dichloride in the presence of anaprotic solvent such as dichloromethane (DCM).

It is further contemplated that these approaches for covalently linkinga polysaccharide to a retinoid through an ester linkage can be extendedto prepare additional non-retinoid polysaccharide therapeutic conjugatecompositions for use where sustained delivery of a therapeutic agent isdesirable.

The polysaccharide retinoid conjugates described herein can beadministered as prodrugs to give a sustained release of the activeretinoid over time. Advantages thereof include a decrease in toxicityeffects of the free retinoid, economizing of the amount of retinoidneeded due to an increase in circulation time and facilitatingsolubilization of hydrophobic retinoids. The particular polysaccharideand molecular weight thereof can be selected to suit the particularapplication, of which retinopathy therapeutic applications are ofparticular interest.

Thus, in another embodiment of the application, a method for treating anocular disorder or ophthalmic disease in a subject includesadministering to the subject a therapeutically effective amount of acomposition that includes a polysaccharide and at least one retinoid.The at least one retinoid can be linked to at least one monosaccharidesubunit of the polysaccharide with a covalent linkage. The linkage canbe degradable by hydrolysis during digestion of the composition toprovide sustained delivery of the retinoid upon enteral administrationof the composition to a subject.

In some embodiments, a therapeutic polysaccharide conjugate compositiondescribed herein can be administered to a subject for treating, curing,preventing, ameliorating the symptoms of, or slowing, inhibiting, orstopping the progression of ocular or ophthalmic diseases or disorders.Representative ophthalmic diseases and disorders include, but are notlimited to, macular degeneration, glaucoma, diabetic retinopathy,retinal detachment, retinal blood vessel occlusion, retinitispigmentosa, autosomal dominant retinitis pigmentosa (ADRP), opticneuropathy, inflammatory retinal disease, diabetic maculopathy,hemorrhagic retinopathy, retinopathy of prematurity, optic neuropathy,proliferative vitreoretinopathy, retinal dystrophy, ischemia-reperfusionrelated retinal injury, hereditary optic neuropathy, metabolic opticneuropathy, Leber congenital amaurosis (LCA) including LCA arising frommutations in the LRAT and RPE65 genes, Stargardt's macular dystrophy,Sorsby's fundus dystrophy, Fundus albipunctatus, congenital stationarynightblindness (CSNB), Best disease, uveitis, age-related retinaldysfunction, a retinal injury, a retinal disorder associated withParkinson's disease, a retinal disorder associated with viral infection,a retinal disorder related to light overexposure, and a retinal disorderassociated with AIDS, a retinal disorder associated with Alzheimer'sdisease, and a retinal disorder associated with multiple sclerosis.

Vitamin A, retinol, plays essential roles in many biological processesincluding vision, immunity, growth, development, and cellulardifferentiation. Therefore, in some embodiments, the polysaccharideretinoid conjugates described herein can be administered to a subjectfor retinoid replacement, supplementing the levels of endogenousretinoid. For example, a polysaccharide retinoid conjugate of thepresent invention can be administered to a subject having a vitamin Adeficiency (VAD). In some embodiments, a polysaccharide retinoidconjugate described herein can be administered to a subject for thepharmacological inhibition of the retinoid cycle.

The polysaccharide retinoid conjugates can be converted directly orindirectly into a retinal or a synthetic retinal analog by degrading thecovalent linkage between the polysaccharide and the retinoid duringdigestion. Thus, in some embodiments, the compositions can be describedas a pro-drug, which upon enteral administration and metabolictransformation is converted into a therapeutic retinoid such as9-cis-retinal, 11-cis-retinal or a synthetic retinal analog thereof.Metabolic transformation can occur, for example by acid hydrolysis,esterase activity, acetyltransferase activity, dehydrogenase activity,or the like. In some embodiments, the compositions described herein whenadministered to a subject enterally allow for longer persistence in thedigestive track and sustained delivery or sustained plasma level.

In some embodiments, methods of using a polysaccharide retinoidconjugate are provided to restore or stabilize photoreceptor function,or to ameliorate photoreceptor loss, in a vertebrate visual system. Asynthetic retinal derivative can be administered to a subject having aretinoid deficiency (e.g., a deficiency of 11-cis-retinal), an excess offree opsin, an excess of retinoid waste (e.g., degradation) products orintermediates in the recycling of all-trans-retinal, or the like. Thevertebrate eye typically comprises a wild-type opsin protein. Methods ofdetermining endogenous retinoid levels in a vertebrate eye, and adeficiency of such retinoids, are disclosed in, for example, U.S.Provisional Patent Application No. 60/538,051 (filed Feb. 12, 2004) (thedisclosure of which is incorporated by reference herein). Other methodsof determining endogenous retinoid levels in a vertebrate eye, and adeficiency of such retinoids, include for example, analysis by highpressure liquid chromatography (HPLC) of retinoids in a sample from asubject. For example, retinoid levels or a deficiency in such levels canbe determined from a blood sample from a subject.

A blood sample can be obtained from a subject and retinoid types andlevels in the sample can be separated and analyzed by normal phase highpressure liquid chromatography (HPLC) (e.g., with a Agilent HP1100 HPLCand a Beckman, Ultrasphere-Si, 4.6 mm×250 mm column using 10% ethylacetate/90% hexane at a flow rate of 1.4 ml/minute). The retinoids canbe detected by, for example, detection at 325 nm using a diode-arraydetector and HP ChemstationA.03.03 software. A deficiency in retinoidscan be determined, for example, by comparison of the profile ofretinoids in the sample with a sample from a control subject (e.g., anormal subject). As used herein, absent, deficient or depleted levels ofendogenous retinoid, such as 11-cis-retinal, refer to levels ofendogenous retinoid lower than those found in a healthy eye of avertebrate of the same species.

The polysaccharide retinoid conjugates can be administeredprophylactically or therapeutically to a vertebrate. As used herein,“prophylactic” and “prophylactically” refer to the administration of apolysaccharide retinoid conjugate to prevent deterioration or furtherdeterioration of the vertebrate visual system, as compared with acomparable vertebrate visual system not receiving the synthetic retinalderivative.

The term “restore” refers to a long-term (e.g., as measured in weeks ormonths) improvement in photoreceptor function in a vertebrate visualsystem, as compared with a comparable vertebrate visual system notreceiving the polysaccharide retinoid conjugate. The term “stabilize”refers to minimization of additional degradation in a vertebrate visualsystem, as compared with a comparable vertebrate visual system notreceiving the polysaccharide retinoid conjugate.

In one embodiment, the subject is characterized as having LeberCongenital Amaurosis (“LCA”). This disease is a very rare childhoodcondition that effects children from birth or shortly thereafter. Itaffects both rods and cones in the eye. For example, certain mutationsin the genes encoding RPE65 and LRAT proteins are involved in LCA.Mutations in both genes result in a person's inability to make11-cis-retinal in adequate quantities. Thus, 11-cis-retinal is eitherabsent or present in reduced quantities. In RPE65-defective individuals,retinyl esters build up in the RPE. LRAT-defective individuals areunable to make esters and subsequently secrete any excess retinoids. ForLCA, a polysaccharide retinoid conjugate can be used to replace theabsent or depleted 11-cis-retinal.

In another embodiment, the vertebrate eye is characterized as havingRetinitis Punctata Albesciens. This disease is a form of RetinitisPigmentosa that exhibits a shortage of 11-cis-retinal in the rods. Apolysaccharide retinoid conjugate can be used to replace the absent ordepleted 11-cis retinal.

In another embodiment, the vertebrate eye is characterized as havingCongenital Stationary Night Blindness (“CSNB”) or Fundus Albipunctatus.This group of diseases is manifested by night blindness, but there isnot a progressive loss of vision as in the Retinitis Pigmentosa. Someforms of CSNB are due to a delay in the recycling of 11-cis-retinal.Fundus Albipunctatus until recently was thought to be a special case ofCSNB where the retinal appearance is abnormal with hundreds of smallwhite dots appearing in the retina. It has been shown that this is alsoa progressive disease, although with a much slower progression thanRetinitis Pigmentosa. It is caused by a gene defect that leads to adelay in the cycling of 11-cis-retinal. Thus, a polysaccharide retinoidconjugate(s) can be administered to restore photoreceptor function byretinoid replacement.

In yet another embodiment, the vertebrate eye is characterized as havingage-related macular degeneration (“AMD”). AMD can be wet or dry forms.In AMD, vision loss occurs when complications late in the disease eithercause new blood vessels to grow under the retina or the retinaatrophies. Without intending to be bound by any particular theory,excessive production of waste products from the photoreceptors mayoverload the RPE. This is due to a shortfall of 11-cis-retinal availableto bind opsin. Free opsin is not a stable compound and can spontaneouslycause firing of the biochemical reactions of the visual cascade withoutthe addition of light.

Administration of a polysaccharide retinoid conjugate to the vertebrateeye can reduce the deficiency of 11-cis-retinal and quench spontaneousmisfiring of the opsin. Administration of a polysaccharide retinoidconjugate can lessen the production of waste products and/or lessendrusen formation, and reduce or slow vision loss (e.g., choroidalneovascularization and/or chorioretinal atrophy).

In other embodiments, a polysaccharide retinoid conjugate isadministered to an aging subject, such as a human. As used herein, anaging human subject is typically at least 45, or at least 50, or atleast 60, or at least 65 years old. The subject has an aging eye, whichis characterized as having a decrease in night vision and/or contrastsensitivity. Excess unbound opsin randomly excites the visualtransduction system. This creates noise in the system and thus morelight and more contrast are necessary to see well. Quenching these freeopsin molecules with a therapeutic retinoid disassociated from apolysaccharide retinoid conjugate will reduce spontaneous misfiring andincrease the signal to noise ratio, thereby improving night vision andcontrast sensitivity.

The subject can include vertebrates, such as, human and non-humanvertebrates. Examples of non-human vertebrates include mammals, such asdogs (canine), cats (feline), horses (equine) and other domesticatedanimals.

The polysaccharide retinoid conjugates can be substantially pure, inthat it contains less than about 5% or less than about 1%, or less thanabout 0.1%, of other retinoids. In some embodiments, a combination ofpolysaccharide retinoid conjugates described herein can be administeredto a subject.

Polysaccharide retinoid conjugates can be formulated, for example, aspharmaceutical compositions for oral administration, localadministration to the eye and/or for intravenous, intramuscularadministration.

Polysaccharide retinoid conjugates can be formulated for administrationusing pharmaceutically acceptable vehicles as well as techniquesroutinely used in the art. A vehicle can be selected according to thesolubility of the polysaccharide retinoid conjugate. Examples ofpharmaceutical compositions include those that are administrableenterally or orally.

Examples of oral dosage forms include tablets, pills, sachets, orcapsules of hard or soft gelatin, methylcellulose or of another materialeasily dissolved in the digestive tract. Examples of nontoxic solidcarriers can include pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like. (See, e.g., Remington“Pharmaceutical Sciences”, 17 Ed., Gennaro (ed.), Mack Publishing Co.,Easton, Pa. (1985).)

The doses of the polysaccharide retinoid conjugates can be selecteddepending on the clinical status, condition and age of the subject,dosage form and the like.

For example, oral doses can typically range from about 0.01 to about1000 mg, one to four times, or more, per day, week, or month. Anexemplary dosing range for oral administration is from about 10 to about250 mg one to three times per week.

Example

We designed a polysaccharide therapeutic conjugate composition for thesustained release of 9-cis-retinyl esters to treat animal models ofhuman retinal diseases, such as LCA. We found that administration of9-cis-R-Ac by oral gavage improves visual function and preserves retinalmorphology in LCA mouse models with LRAT and RPE65 deficiency, that is,Lrat^(−/−) and Rpe65^(−/−) mice. We also found that 9-cis-retinoidconcentrations in plasma increased rapidly to high levels after gavageand then dropped markedly to low levels 5 hours after oraladministration. Unless initial high drug blood levels are unfavorable,this pharmacokinetic property could be beneficial because it relates tothe therapeutic window, especially for drugs targeting peripheraltissues such as the retina through protein-mediated andprotein-independent transport systems. A bolus dose could also establisha reversible depot of the drug in other tissues, thereby reducing theneed for frequent dosing. For example, we observed that a single dose of9-cis-retinal to Rpe65^(−/−) mice rescued visual function for months,even when animals were kept under laboratory lighting conditions.

Nonetheless, sustained therapeutic dosing methods can hold numerousadvantages over bolus dosing, including a lower risk of toxicity and anincreased duration of therapeutic efficacy. In the case of retinoidsupplementation therapy, it provides an additional source for the drug.The retina poses unique challenges for sustained therapeutic dosingbecause of its anatomic isolation. Frequent injections of compoundsdirectly into the vitreous cavity are associated with complications,such as retinal detachment, hemorrhage, uveitis, endophthalmitis, andinfections. Therefore, delivery of hydrophobic 9-cis-retinoids directlyby this route presents an additional concern.

The Lrat^(−/−) mouse provides an excellent model for human LCA becauseit exhibits early-onset, slowly progressive severe retinal degeneration.Because the RPE of Lrat^(−/−) mice is devoid of all-trans-retinol andall-trans-retinyl esters, the photoreceptors lack functional rhodopsin,and electroretinographic (ERG) responses are dramatically attenuated.Lrat^(−/−) mice have been used to test the efficacy of pharmacologicagents such as 9-cis-R-Ac and a standard gene replacement technique.Moreover, lack of LRAT in the liver and other tissues has no apparentdeleterious effects. These mice constitute an important experimentalmodel for retinoid metabolism and vitamin A deprivation as well as forhuman LCA.

To achieve sustained release of 9-cis-retinoids, we designed apolysaccharide therapeutic conjugate composition that uses a chitosan,which is covalently linked to 9-cis-retinoids. The covalent linkage ishydrolyzable upon enteral administration to provide sustained deliveryof the 9-cis-retinoids to the eye of subject at least 24 hours (e.g., 24hours to 1 week) after enteral administration. The polysaccharidetherapeutic conjugate composition slows drug release upon enteraladministration and thus blunts an initial excessive drug release. Thisdrug-delivery system can maintain optimal therapeutic drug levels,reduce side effects by sustained drug release, and improve patientcompliance by decreasing the frequency of drug administration in aminimally invasive manner. To show sustained retinoid release could beachieved by using a polysaccharide therapeutic conjugate composition andrescue visual function, we prepared a chitosan-retinoid conjugate asshown in FIG. 6, investigated 9-cis-retinoid release from thiscomposition in vivo, and evaluated its therapeutic efficacy in retinasof Lrat^(−/−) mice.

Animals

Lrat−/− mice were generated and genotyped and 5-week-old animals of bothsexes were used for the experiments described. All mice were housed inthe animal facility at the School of Medicine, Case Western ReserveUniversity, where they were maintained on a standard diet under a12-hour light (<10 lux)/12-hour dark cycle. At least 24 hours beforeexperiments were initiated, mice were placed and maintained in a darkenvironment. Procedures were performed under dim red light transmittedthrough a safelight filter (transmittance >560 nm; No. 1; Kodak,Rochester, N.Y.). All animal procedures and experiments were approved bythe Case Western Reserve University Animal Care Committee and conformedto recommendations of both the American Veterinary Medical AssociationPanel on Euthanasia and the ARVO Statement for the Use of Animals inOphthalmic and Vision Research.

Retinoid Administration

All chitosan-retinoid compositions were administrated to Lrat−/− miceunder dim red light. The chitosan-retinoid compositions were orallygavaged with soybean oil (95%, v/v) to a final concentration of 20mg/mL. The soybean oil preparation (300 μL) was gavaged into mice as asingle dose of 0.012 mg of retinoid/mouse.

Electroretinography (ERG) Recording

Lrat^(−/−) mice were dark-adapted prior to the procedure and thenanesthetized by intraperitoneal injection of a mixture containingketamine (80 mg/kg body weight) and xylazine (20 mg/kg body weight) in10 mM sodium phosphate, pH 7.2, with 100 mM NaCl. Pupils were dilatedwith 1 drop of 1% tropicamide ophthalmic solution. ERG responses weremeasured with a universal testing and electrophysiologic system (UTASwith BigShot; LKC Technologies, Inc., Gaithersburg, Md.). To stabilizebody temperature, mice were placed on a heated pad (37° C.) in aGanzfeld chamber, where positioning of the contact lens electrodes wasadjusted. Then two needle electrodes were placed under the skin of theforehead and tail as a reference and ground electrode, respectively, andtwo contact lens electrodes were placed on the eyes. Recording commencedwhen the lenses were well contacted and the baseline was stable.Single-flash ERGs were recorded simultaneously from both eyes withscotopic flash intensities (from −3.7 to 1.6 log cd sm⁻²), and two tofive recordings were made at sufficient intervals between flash stimuli(from 10-60 seconds) to allow mice time to recover. Amplitudes weremeasured by a commercial software package (EM software for Windows; LKCTechnologies, Inc.) from baseline to the negative peak for a-waves andfrom the trough to the highest peak for b-waves.

Retinoid Analyses

All experimental procedures related to extraction, derivatization, andseparation of retinoids were performed under dim red light provided by aKodak No. 1 safelight filter (transmittance >560 nm). Retinoids wereextracted twice with an equal volume of 100% hexane (6 mL total). Thecombined extracts were dried under argon, and retinoids were separatedon a normal-phase HPLC column (Ultrasphere-Si, 5 μm, 4.6×250 mm;Beckman) with 10% ethyl acetate and 90% hexane at a flow rate of 1.4mL/min and detected at 325 nm by an HPLC system (model HP1100; HewlettPackard, with a diode array detector and HP Chemstation A.03.03software).

Histologic Evaluation

Briefly, mouse eyecups were prepared immediately after euthanasia byremoval of the cornea and lens. Eyecups were fixed in 2% glutaraldehyde,4% paraformaldehyde and processed for embedding in Epon. Sections forroutine histology were cut at 1 lm and stained with toluidine blue.

HPLC Analysis of Eyes after Oral Gavage of Chitosan 9-Cis-RetinolConjugate

To confirm whether orally gavaged chitosan 9-cis-retinol conjugates canproduce 9-cis-retinal oximes in the eye, we extracted the retinoids frompurified visual pigment of Lrat−/− mice orally gavaged chitosan9-cis-retinol conjugates and detected 9-cis-retinal oximes. Significantamounts of 9-cis-retinal oximes were detected in eyes of treated miceexposed to intense light 6 hours after oral gavage and 23 hours afteroral gavage (FIGS. 8A-B), whereas the amounts of 9-cis-retinal oximeswere not detected in control groups not administered the therapeutic.

Improved ERG Responses in Lrat^(−/−) Mice 1 Month after Oral Gavage ofChitosan 9-Cis-Retinol Congates

Rod and cone functions of Lrat^(−/−) mice become dramatically attenuatedbecause of chromophore deficiency and resulting absence of visualpigments. Rods of Lrat^(−/−) mice are approximately 2000-fold lesssensitive to light stimuli than WT rods. Delivering the artificial9-cis-retinoid chromophore to the retina leads to generation of thevisual pigment, isorhodopsin, in Lrat^(−/−) mice, thereby restoringvisual function. We recorded ERG responses in Lrat^(−/−) mice treatedwith chitosan 9-cis-retinol conjugates to monitor their visual function.All experiments were initiated with 5-week-old Lrat^(−/−) mice. Chitosan9-cis-retinol conjugates were orally administered to Lrat^(−/−) mice in6 oral gavages of 0.012 mg/mouse (n=9) over 2 weeks. All mice weremaintained under a regular 12-hour light (<10 lux)/12-hour dark cycle.Three weeks after drug administration, mice were transferred to adarkroom for 1 week, and then single-flash scotopic ERGs were recorded.FIGS. 7 A-B show delivery of the oral gavage of chitosan 9-cis-retinolconjugates produced significant increases in both a-wave and b-waveamplitudes, starting at a stimulus intensity of 1.6 log cd·s·m⁻² fora-waves and 0.4 log cd·s·m⁻² for b-waves compared to baseline responsesin untreated Lrat^(−/−) mice. Thus, the increase of both ERG a-wave andb-wave amplitudes suggested that release of 9-cis-retinol from thechitosan 9-cis-retinol conjugates was sustained continuously asindicated in the retinoid-releasing profile. This maintained release of9-cis-retinol caused an improvement of retinal function in Lrat^(−/−)mice at even 4 weeks after administration, which was not attainable bysingle dose oral treatment.

Retinal Histology of 9-cis-R-Ac-Treated Lrat^(−/−) Mice

Functional rhodopsin (or isorhodopsin) is required to maintain normalROS morphology. Therefore, if treatment of Lrat^(−/−) mice with9-cis-retinoids can restore visual function, it should also preserve ROSstructure. Histologic analyses revealed that at the age of 6-8 weeks,ROS lengths of Lrat^(−/−) mice were reduced to 65% of those observed inWT control mice. EM analysis of retinas demonstrated that ROS layers ofLrat^(−/−) mice were shorter, thinner, and less tightly packed thanthose of WT mice. It was also reported that, after gavaging dark roomreared Lrat^(−/−) mice with a high dose of 9-cis-R-Ac (total dose of 72mg in 6 gavages of 0.12 mg each over a two week duration), the ROS layerbecame substantially thicker, and the RPE-ROS interface displayed closerapposition than that seen in untreated control Lrat^(−/−) mice. In thisstudy, we observed histologic retention of ROS structures 1 month afteroral gavage of chitosan 9-cis-retinol conjugates (FIGS. 9A-B). The ROSlayer of the subcutaneous was thicker than that of the untreated controlgroup, and more tightly packed than ROS layers untreated control groups.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications Such improvements,changes and modifications are within the skill of the art and areintended to be covered by the appended claims. All publications,patents, and patent applications cited in the present application areherein incorporated by reference in their entirety.

Having described the invention, the following is claimed:
 1. Acomposition consisting of: a polysaccharide; and at least one retinoidlinked to at least one monosaccharide subunit of the polysaccharide toform a hydrolysable carboxylic ester linkage, the linkage beingdegradable by hydrolysis during digestion of the composition to providecontrolled, delayed, and/or sustained delivery of at least one retinolupon enteral administration of the composition to a subject, thepolysaccharide comprising chitosan, and wherein the at least oneretinoid comprises a retinal derivative.
 2. The composition of claim 1,wherein the carboxylic ester linkage is a linker that connects themonosaccharide subunit of the polysaccharide to the retinoid, the linkerhaving two or more functional groups for forming hydrolysable covalentbonds with the at least one monomer subunit of the polysaccharide andwith the retinoid.
 3. The composition of claim 1, wherein the retinoidcomprises a synthetic retinoid of formula I, II, III, IV, V, VI, VII,VIII, IX, X, XI, XII, XIII, XIV, XV or XVI.
 4. The composition of claim1, wherein the retinoid comprises 9-cis retinol or a derivative thereoffor forming the hydrolysable carboxylic ester linkage.
 5. Thecomposition of claim 1, wherein the retinoid comprises 11-cis retinol ora derivative thereof for forming the hydrolysable carboxylic esterlinkage.
 6. The composition of claim 1, wherein the retinoid comprises aterminal alkyl halide.
 7. The composition of claim 6, wherein theterminal alkyl halide comprises a terminal alkyl chloride.
 8. Thecomposition of claim 7, wherein the retinoid comprises 9-cis retinalchloride.
 9. A composition consisting of: a polysaccharide; and at leastone retinoid linked to at least one monosaccharide subunit of thepolysaccharide to form a hydrolysable carboxylic ester linkage, thelinkage being degradable by hydrolysis during digestion of thecomposition to provide controlled, delayed, and/or sustained delivery ofat least one retinol upon enteral administration of the composition to asubject, wherein the monosaccharide subunit comprises at least one ofβ-D-mannuronate (M) or α-L-guluronate (G) and the at least one retinoidcomprises a retinal derivative.
 10. The composition of claim 9, whereinthe retinoid comprises a 9-cis retinoid.
 11. A composition consistingof: a polysaccharide; and at least one 9-cis retinol or derivativethereof linked to at least one monosaccharide subunit of thepolysaccharide to form a hydrolysable covalent linkage, the linkagebeing degradable by hydrolysis during digestion of the composition toprovide controlled, delayed, and/or sustained delivery of at least one9-cis retinol upon enteral administration of the composition to asubject.
 12. The composition of claim 9, wherein the at least oneretinoid is directly linked to the at least one monosaccharide subunitof the polysaccharide through the carboxylic ester linkage.
 13. Thecomposition of claim 9, wherein the carboxylic ester linkage is a linkerthat connects the monosaccharide subunit of the polysaccharide to theretinoid, the linker having two or more functional groups for forminghydrolysable covalent bonds with the at least one monomer subunit of thepolysaccharide and with the retinoid.
 14. The composition of claim 9,wherein the retinoid comprises a synthetic retinoid of formula I, II,III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI.
 15. Thecomposition of claim 9, wherein the retinoid comprises 9-cis retinol ora derivative thereof.
 16. The composition of claim 9, wherein theretinoid comprises 11-cis retinol or a derivative thereof.
 17. Thecomposition of claim 9, wherein the retinoid comprises a terminal alkylhalide.
 18. The composition of claim 17, wherein the terminal alkylhalide comprises a terminal alkyl chloride.
 19. The composition of claim18, wherein the retinoid comprises 9-cis retinal chloride.
 20. Thecomposition of claim 11, wherein the hydrolysable covalent linkage isselected from the group consisting of ester, ether, thioether,disulfide, amide, imide, secondary amine, direct carbon (C—C),carboxylic ester, sulfate ester, sulfonate ester, phosphate ester,urethane and carbonate hydrolysable covalent linkages.
 21. Thecomposition of claim 11, wherein the hydrolysable covalent linkagecomprises a hydrolysable carboxylic ester linkage.
 22. The compositionof claim 11, wherein the at least one monosaccharide subunit of thepolysaccharide has a carboxylate ion or amine residue, the carboxylateion residue or amine residue forming the covalent linkage with the 9-cisretinol or derivative thereof.
 23. The composition of claim 11, whereinthe at least one 9-cis retinol or derivative thereof is directly linkedto the at least one monosaccharide subunit of the polysaccharide throughthe covalent linkage.
 24. The composition of claim 11, wherein thecovalent linkage is a linker that connects the monosaccharide subunit ofthe polysaccharide to the 9-cis retinol or derivative thereof, thelinker having two or more functional groups for forming hydrolysablecovalent bonds with the at least one monomer subunit of thepolysaccharide and with the 9-cis retinol or derivative thereof.
 25. Thecomposition of claim 11, wherein the at least one monosaccharide subunitof the polysaccharide is selected from the group consisting ofβ-D-mannuronate (M) and α-L-guluronate (G).
 26. The composition of claim11, wherein the polysaccharide comprises chitosan.
 27. The compositionof claim 9, wherein the hydrolysable carboxylic ester linkage has astructure of:


28. The composition of claim 21, wherein the hydrolysable carboxylicester linkage has a structure of: